Genetically modified major histocompatibility complex mice

ABSTRACT

The invention provides genetically modified non-human animals that express a humanized MHC II protein (humanized MHC II α and β polypeptides), as well as embryos, cells, and tissues comprising the same. Also provided are constructs for making said genetically modified animals and methods of making the same. Methods of using the genetically modified animals to study various aspects of human immune system are provided.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims benefit of priority to U.S. ProvisionalApplication No. 61/552,584, filed Oct. 28, 2011, which is incorporatedby reference herein in its entirety.

FIELD OF THE INVENTION

Present invention relates to a non-human animal, e.g., a rodent (e.g., amouse or a rat) that is genetically engineered to express a humanizedMajor Histocompatibility Complex (MHC) class II protein, as well asembryos, tissues, and cells expressing the same. The invention furtherrelates to methods for making a genetically modified non-human animalthat expresses a humanized MHC II protein. Also provided are methods forusing non-human animals, cells, and tissues that express a humanized MHCclass II protein for identifying peptides that activate lymphocytes andengage T cells, and for developing human vaccines and othertherapeutics.

BACKGROUND OF THE INVENTION

In the adaptive immune response, foreign antigens are recognized byreceptor molecules on B lymphocytes (e.g., immunoglobulins) and Tlymphocytes (e.g., T cell receptor or TCR). These foreign antigens arepresented on the surface of cells as peptide fragments by specializedproteins, generically referred to as major histocompatibility complex(MHC) molecules. MHC molecules are encoded by multiple loci that arefound as a linked cluster of genes that spans about 4 Mb. In mice, theMHC genes are found on chromosome 17, and for historical reasons arereferred to as the histocompatibility 2 (H-2) genes. In humans, thegenes are found on chromosome 6 and are called human leukocyte antigen(HLA) genes. The loci in mice and humans are polygenic; they includethree highly polymorphic classes of MHC genes (class I, II and III) thatexhibit similar organization in human and murine genomes (see FIG. 2 andFIG. 3, respectively).

MHC loci exhibit the highest polymorphism in the genome; some genes arerepresented by >300 alleles (e.g., human HLA-DRβ and human HLA-B). Allclass I and II MHC genes can present peptide fragments, but each geneexpresses a protein with different binding characteristics, reflectingpolymorphisms and allelic variants. Any given individual has a uniquerange of peptide fragments that can be presented on the cell surface toB and T cells in the course of an immune response.

Both humans and mice have class II MHC genes (see FIGS. 2 and 3). Inhumans, the classical MHC II genes are termed HLA-DP, HLA-DQ, andHLA-DR, whereas in mice they are H-2A and H-2E (often abbreviated as I-Aand I-E, respectively). Additional proteins encoded by genes in the MHCII locus, HLA-DM and HLA-DO in humans, and H-2M and H-20 in mice, arenot found on the cell surface, but reside in the endocytic compartmentand ensure proper loading of MHC II molecules with peptides. Class IImolecules consist of two polypeptide chains: α chain and β chain. Theextracellular portion of the α chain contains two extracellular domains,α1 and α2; and the extracellular portion of the β chain also containstwo extracellular domains, β1 and β2 (see FIG. 1). The α and the βchains are non-covalently associated with each other.

MHC class II molecules are expressed on antigen-presenting cells (APCs),e.g., B cells, macrophages, dendritic cells, endothelial cells during acourse of inflammation, etc. MHC II molecules expressed on the surfaceof APCs typically present antigens generated in intracellular vesiclesto CD4+ T cells. In order to participate in CD4+ T cell engagement, theMHC class II complex with the antigen of interest must be sufficientlystable to survive long enough to engage a CD4+ T cell. When a CD4+ Thelper cell is engaged by a foreign peptide/MHC II complex on thesurface of APC, the T cell is activated to release cytokines that assistin immune response to the invader.

Not all antigens will provoke T cell activation due to tolerancemechanisms. However, in some diseases (e.g., cancer, autoimmunediseases) peptides derived from self-proteins become the target of thecellular component of the immune system, which results in destruction ofcells presenting such peptides. There has been significant advancementin recognizing antigens that are clinically significant (e.g., antigensassociated with various types of cancer). However, in order to improveidentification and selection of peptides that will provoke a suitableresponse in a human T cell, in particular for peptides of clinicallysignificant antigens, there remains a need for in vivo and in vitrosystems that mimic aspects of human immune system. Thus, there is a needfor biological systems (e.g., genetically modified non-human animals andcells) that can display components of a human immune system.

SUMMARY OF THE INVENTION

A biological system for generating or identifying peptides thatassociate with human MHC class II proteins and chimeras thereof, andbind to CD4+ T cells, is provided. Non-human animals comprisingnon-human cells that express humanized molecules that function in thecellular immune response are provided. Humanized rodent loci that encodehumanized MHC II proteins are also provided. Humanized rodent cells thatexpress humanized MHC molecules are also provided. In vivo and in vitrosystems are provided that comprise humanized rodent cells, wherein therodent cells express one or more humanized immune system molecules.

Provided herein is a non-human animal, e.g., a rodent (e.g., a mouse ora rat) comprising in its genome a nucleotide sequence encoding ahumanized MHC II complex, wherein a human portion of the humanized MHCII complex comprises an extracellular domain of a human MHC II complex,e.g., a humanized MHC II α extracellular domain and a humanized MHC II βextracellular domain.

In one aspect, provided herein is a non-human animal comprising at anendogenous MHC II α gene locus a nucleotide sequence encoding a chimerichuman/non-human MHC II α polypeptide. In one embodiment, a human portionof such chimeric human/non-human MHC II α polypeptide comprises a humanMHC II α extracellular domain. In one embodiment, the non-human animalexpresses a functional MHC II complex on a surface of a cell of theanimal. In one embodiment, the human MHC II α extracellular domain inthe animal comprises human MHC II α1 and α2 domains; in one embodiment,a non-human portion of the chimeric human/non-human MHC II α polypeptidecomprises transmembrane and cytoplasmic domains of an endogenousnon-human MHC II α polypeptide. In one embodiment, the nucleotidesequence encoding a chimeric human/non-human MHC II α polypeptide isexpressed under regulatory control of endogenous non-human MHC II αpromoter and regulatory elements. In one embodiment, the human portionof the chimeric polypeptide is derived from a human HLA class II proteinselected from the group consisting of HLA-DR, HLA-DQ, and HLA-DP, e.g.,the human portion is derived from HLA-DR4 protein. The non-human animalmay be a rodent, e.g., a mouse. In one aspect, the non-human animalcomprising at an endogenous MHC II α gene locus a nucleotide sequenceencoding a chimeric human/non-human MHC II α polypeptide furthercomprises at an endogenous MHC II β gene locus a nucleotide sequenceencoding a chimeric human/non-human MHC II β polypeptide. Also providedherein is a method of making a genetically modified non-human animalcomprising at an endogenous MHC II α gene locus a nucleotide sequenceencoding a chimeric human/non-human MHC II α polypeptide. Such methodmay comprise replacing at an endogenous MHC II α gene locus a nucleotidesequence encoding an endogenous non-human MHC II α polypeptide with anucleotide sequence encoding a chimeric human/non-human MHC II αpolypeptide.

Also provided herein is a non-human animal comprising at an endogenousMHC II β gene locus a nucleotide sequence encoding a chimerichuman/non-human MHC II β polypeptide. In one embodiment, a human portionof such chimeric human/non-human MHC II β polypeptide comprises a humanMHC II β extracellular domain. In one embodiment, the non-human animalexpresses a functional MHC II complex on a surface of a cell of theanimal. In one embodiment, the human MHC II β extracellular domain inthe animal comprises human MHC II β1 and β2 domains; in one embodiment,a non-human portion of the chimeric human/non-human MHC II β polypeptidecomprises transmembrane and cytoplasmic domains of an endogenousnon-human MHC II β polypeptide. In one embodiment, the nucleotidesequence encoding a chimeric human/non-human MHC II β polypeptide isexpressed under regulatory control of endogenous non-human MHC II βpromoter and regulatory elements. In one embodiment, the human portionof the chimeric polypeptide is derived from a human HLA class II proteinselected from the group consisting of HLA-DR, HLA-DQ, and HLA-DP, e.g.,the human portion is derived from HLA-DR4 protein. The non-human animalmay be a rodent, e.g., a mouse. In one aspect, the non-human animalcomprising at an endogenous MHC II β gene locus a nucleotide sequenceencoding a chimeric human/non-human MHC II β polypeptide furthercomprises at an endogenous MHC II α gene locus a nucleotide sequenceencoding a chimeric human/non-human MHC II α polypeptide. Also providedherein is a method of making a genetically modified non-human animalcomprising at an endogenous MHC II β gene locus a nucleotide sequenceencoding a chimeric human/non-human MHC II β polypeptide. Such methodmay comprise replacing at an endogenous MHC II β gene locus a nucleotidesequence encoding an endogenous non-human MHC II β polypeptide with anucleotide sequence encoding a chimeric human/non-human MHC II βpolypeptide.

In one aspect, a non-human animal is provided comprising at anendogenous MHC II gene locus a first nucleotide sequence encoding achimeric human/non-human MHC II α polypeptide and a second nucleotidesequence encoding a chimeric human/non-human MHC II β polypeptide,wherein a human portion of the chimeric human/non-human MHC II αpolypeptide comprises a human MHC II α extracellular domain and a humanportion of the chimeric human/non-human MHC II β polypeptide comprises ahuman MHC II β extracellular domain. In one embodiment, the chimerichuman/non-human MHC II α and β polypeptides form a functional chimericMHC II complex (e.g., human/non-human MHC II complex) on a surface of acell. In one embodiment, the human MHC II α extracellular domaincomprises human α1 and α2 domains of human MHC II. In one embodiment,the human MHC II β extracellular domain comprises human β1 and β2domains of human MHC II. In various aspects, the first nucleotidesequence is expressed under regulatory control of endogenous non-humanMHC II α promoter and regulatory elements. In various aspects, thesecond nucleotide sequence is expressed under regulatory control ofendogenous non-human MHC II β promoter and regulatory elements. In someembodiments, a non-human portion of the chimeric human/non-human MHC IIα polypeptide comprises transmembrane and cytoplasmic domains of anendogenous non-human MHC II α polypeptide. In some embodiments, anon-human portion of the chimeric human/non-human MHC II β polypeptidecomprises transmembrane and cytoplasmic domains of an endogenousnon-human MHC II β polypeptide.

In various embodiments, the non-human animal is a rodent, and the humanportions of the chimeric human/rodent MHC II α and β polypeptidescomprise human sequences derived from HLA class II protein selected fromthe group consisting of HLA-DR, HLA-DQ, and HLA-DP. In some embodimentsof the invention, the human portions of the chimeric human/rodent MHC IIα and β sequences are derived from a human HLA-DR4 sequence; thus, thenucleotide sequence encoding the MHC II α extracellular domain isderived from a sequence of an HLA-DRα*01 gene, and the nucleotidesequence encoding the MHC II β extracellular domain is derived from asequence encoding an HLA-DRβ1*04 gene.

In various embodiments of the invention, the first and the secondnucleotide sequences are located on the same chromosome. In someaspects, the animal comprises two copies of the MHC II locus containingthe first and the second nucleotide sequences, while in other aspects,the animal comprises one copy of the MHC II locus containing the firstand the second nucleotide sequences. Thus, the animal may be homozygousor heterozygous for the MHC II locus containing the first and the secondnucleotide sequences.

In some aspects, the chimeric MHC II α polypeptide and/or the chimericMHC II β polypeptide is operably linked to a non-human leader sequence.

In one aspect, the genetically engineered non-human animal is a rodent.In one embodiment, the rodent is selected from the group consisting of amouse and a rat. Thus, in some embodiments, non-human sequences of thechimeric MHC II α and β genes are derived from nucleotide sequencesencoding mouse MHC II protein, e.g., a mouse H-2E protein. In oneembodiment, the rodent (e.g., the mouse or the rat) of the inventiondoes not express functional endogenous MHC II polypeptides from theirendogenous loci. In one embodiment, wherein the rodent is a mouse, themouse does not express functional endogenous H-2E and H-2A polypeptidesfrom their endogenous loci.

Thus, in some embodiments, a mouse is provided comprising at anendogenous mouse MHC II locus a first nucleotide sequence encoding achimeric human/mouse MHC II α polypeptide and a second nucleotidesequence encoding a chimeric human/mouse MHC II β polypeptide, wherein ahuman portion of the chimeric MHC II α polypeptide comprises anextracellular domain derived from an α polypeptide of a human HLA-DR4protein and a human portion of the chimeric human/mouse MHC II βpolypeptide comprises an extracellular domain derived from a βpolypeptide of a human HLA-DR4 protein, wherein a mouse portion of thechimeric MHC II α polypeptide comprises transmembrane and cytoplasmicdomains of a mouse H-2E α chain and a mouse portion of the chimeric MHCII β polypeptide comprises transmembrane and cytoplasmic domains of amouse H-2E β chain, and wherein the mouse expresses a functionalchimeric HLA-DR4/H-2E MHC II complex. In some aspects, the extracellulardomain of the chimeric MHC II α polypeptide comprises human α1 and α2domains; in some aspects, the extracellular domain of the chimeric MHCII β polypeptide comprises human β1 and β2 domains. In some embodiments,the first nucleotide sequence is expressed under regulatory control ofendogenous mouse MHC II α promoter and regulatory elements, and thesecond nucleotide sequence is expressed under regulatory control ofendogenous mouse MHC II β promoter and regulatory elements. In variousembodiments, the mouse does not express functional endogenous MHC IIpolypeptides, e.g., H-2E and H-2A polypeptides, from their endogenousloci. In some aspects, the mouse comprises two copies of the MHC IIlocus containing the first and the second nucleotide sequences, while inother aspects, the mouse comprises one copy of the MHC II locuscontaining the first and the second nucleotide sequences.

Methods of making genetically engineered non-human animals (e.g.,rodents, e.g., mice or rats) as described herein are also provided. Invarious embodiments, non-human animals (e.g., rodents, e.g., mice orrats) of the invention are made by replacing endogenous MHC II sequenceswith nucleotide sequences encoding chimeric human/non-human (e.g.,human/mouse) MHC II α and β polypeptides. In one embodiment, theinvention provides a method of modifying an MHC II locus of a rodent(e.g., a mouse or a rat) to express a chimeric human/rodent MHC IIcomplex comprising replacing at the endogenous mouse MHC II locus anucleotide sequence encoding a rodent MHC II complex with a nucleotidesequence encoding a chimeric human/rodent MHC II complex. In one aspectof the method, the nucleotide sequence encoding the chimerichuman/rodent MHC II complex comprises a first nucleotide sequenceencoding an extracellular domain of a human MHC II α chain andtransmembrane and cytoplasmic domains of a rodent MHC II α chain and asecond nucleotide sequence encoding an extracellular domain of a humanMHC II β chain and transmembrane and cytoplasmic domains of a rodent MHCII β chain. In some aspects, a rodent portion of the chimeric MHC IIcomplex is derived from a mouse H-2E protein, and a human portion isderived from a human HLA-DR4 protein. In some embodiments, thereplacement of the endogenous MHC II loci described herein is made in asingle ES cell, and the single ES cell is introduced into a rodent(e.g., mouse or rat) embryo to make a genetically modified rodent (e.g.,mouse or rat).

Also provided herein are cells, e.g., isolated antigen-presenting cells,derived from the non-human animals (e.g., rodents, e.g., mice or rats)described herein. Tissues and embryos derived from the non-human animalsdescribed herein are also provided.

Any of the embodiments and aspects described herein can be used inconjunction with one another, unless otherwise indicated or apparentfrom the context. Other embodiments will become apparent to thoseskilled in the art from a review of the ensuing detailed description.The following detailed description includes exemplary representations ofvarious embodiments of the invention, which are not restrictive of theinvention as claimed. The accompanying figures constitute a part of thisspecification and, together with the description, serve only toillustrate embodiments and not to limit the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing of the MHC II class molecule expressed onthe surface of an antigen presenting cell (APC), containing fourdomains: α1, α2, β1, and β2. The gray circle represents a peptide boundin the peptide-binding cleft.

FIG. 2 is a schematic representation (not to scale) of the relativegenomic structure of the human HLA, showing class I, II and III genes.

FIG. 3 is a schematic representation (not to scale) of the relativegenomic structure of the mouse MHC, showing class I, II and III genes.

FIG. 4 (A-D) is a schematic illustration (not to scale) of the strategyfor generating a targeting vector comprising humanized I-E β and I-E α(i.e., H-2Eβ/HLA-DRβ1*04 and H-2Eα/HLA-DRα*01 chimera, respectively). InFIG. 4C, the final humanized MHC II sequence from FIG. 4B is ligatedbetween PI-SceI and I-CeuI restriction sites of the final construct fromFIG. 4A, to generate a construct comprising humanized MHC II and exon 1of I-Eα from BALB/c. Pg=pseudogene; BHR=bacterial homologousrecombination; CM=chloramphenicol; spec=spectinomycin; hyg=hygromycin;neo=neomycin; EP=electroporation. Triangles represent exons, filledtriangles represent mouse exons from C57BL/6 mouse (with the exceptionof hashed triangles, which represent exon 1 of I-Eα from BALB/c mouse)and open triangles represent human exons.

FIG. 5 shows a schematic illustration, not to scale, of MHC class II I-Eand I-A genes, showing knockout of the mouse locus using a hygromycincassette, followed by introduction of a vector comprising a humanizedI-E β and I-E α (i.e., H-2Eβ/HLA-DRβ1*04 and H-2Eα/HLA-DRα*01 chimera,respectively). Open triangles represent human exons; filled trianglesrepresent mouse exons. Probes used for genotyping are encircled.

FIG. 6 shows a schematic illustration, not to scale, of Cre-mediatedremoval of the neomycin cassette of FIG. 5. Open triangles representhuman exons; filled triangles represent mouse exons. Top two strandsrepresent MHC II loci in humanized MHC II heterozygous mouse harboring aneomycin selection cassette, and bottom two strands represent MHC IIloci in humanized MHC II heterozygous mouse with neomycin cassetteremoved.

FIG. 7 shows a schematic comparative illustration, not to scale, ofmouse and human class II loci. Class II genes are represented by boxes,and empty boxes represent pseudogenes. Relative sizes (kb) of variousnucleic acid fragments are included.

FIG. 8, at left panel, is a schematic illustration (not to scale) ofhumanization strategy for the MHC II α chain; in particular, the figureshows a replacement of α1 and α2 domains, encoded by exons 2 and 3 ofMHC II α gene, while retaining mouse transmembrane and cytoplasmic tailsequences. In the humanized locus, the MHC II α leader sequence isderived from the mouse BALB/c strain. The right panel illustrateshumanization of the MHC II β chain; in particular, the figure shows areplacement of β1 and β2 domains, encoded by exons 2 and 3 of MHC II βgene, while retaining the mouse leader and mouse transmembrane andcytoplasmic tail sequences. Top row are all human sequences; middle roware all mouse sequences; bottom row are all humanized sequences, withexons 2 and 3 derived from human HLA-DR genes.

FIG. 9 shows FACS analysis with anti-HLA-DR antibody of B cells from amouse heterozygous for a chimeric HLA-DR4 (neo cassette removed) in thepresence (1681 HET+poly(I:C) or absence (1681HET) of poly(I:C), and awild-type mouse (WT mouse).

DETAILED DESCRIPTION OF THE INVENTION Definitions

The present invention provides genetically modified non-human animals(e.g., mice, rats, rabbits, etc.) that express human or humanized MHC IIpolypeptide; embryos, cells, and tissues comprising the same; methods ofmaking the same; as well as methods of using the same. Unless definedotherwise, all terms and phrases used herein include the meanings thatthe terms and phrases have attained in the art, unless the contrary isclearly indicated or clearly apparent from the context in which the termor phrase is used.

The term “conservative,” when used to describe a conservative amino acidsubstitution, includes substitution of an amino acid residue by anotheramino acid residue having a side chain R group with similar chemicalproperties (e.g., charge or hydrophobicity). Conservative amino acidsubstitutions may be achieved by modifying a nucleotide sequence so asto introduce a nucleotide change that will encode the conservativesubstitution. In general, a conservative amino acid substitution willnot substantially change the functional properties of interest of aprotein, for example, the ability of MHC II to present a peptide ofinterest. Examples of groups of amino acids that have side chains withsimilar chemical properties include aliphatic side chains such asglycine, alanine, valine, leucine, and isoleucine; aliphatic-hydroxylside chains such as serine and threonine; amide-containing side chainssuch as asparagine and glutamine; aromatic side chains such asphenylalanine, tyrosine, and tryptophan; basic side chains such aslysine, arginine, and histidine; acidic side chains such as asparticacid and glutamic acid; and, sulfur-containing side chains such ascysteine and methionine. Conservative amino acids substitution groupsinclude, for example, valine/leucine/isoleucine, phenylalanine/tyrosine,lysine/arginine, alanine/valine, glutamate/aspartate, andasparagine/glutamine. In some embodiments, a conservative amino acidsubstitution can be a substitution of any native residue in a proteinwith alanine, as used in, for example, alanine scanning mutagenesis. Insome embodiments, a conservative substitution is made that has apositive value in the PAM250 log-likelihood matrix disclosed in Gonnetet al. ((1992) Exhaustive Matching of the Entire Protein SequenceDatabase, Science 256:1443-45), hereby incorporated by reference. Insome embodiments, the substitution is a moderately conservativesubstitution wherein the substitution has a nonnegative value in thePAM250 log-likelihood matrix.

Thus, also encompassed by the invention is a genetically modifiednon-human animal whose genome comprises a nucleotide sequence encoding ahuman or humanized MHC II polypeptide, wherein the polypeptide comprisesconservative amino acid substitutions in the amino acid sequencedescribed herein.

One skilled in the art would understand that in addition to the nucleicacid residues encoding a human or humanized MHC II polypeptide describedherein, due to the degeneracy of the genetic code, other nucleic acidsmay encode the polypeptide of the invention. Therefore, in addition to agenetically modified non-human animal that comprises in its genome anucleotide sequence encoding MHC II polypeptide with conservative aminoacid substitutions, a non-human animal whose genome comprises anucleotide sequence that differs from that described herein due to thedegeneracy of the genetic code is also provided.

The term “identity” when used in connection with sequence includesidentity as determined by a number of different algorithms known in theart that can be used to measure nucleotide and/or amino acid sequenceidentity. In some embodiments described herein, identities aredetermined using a ClustalW v. 1.83 (slow) alignment employing an opengap penalty of 10.0, an extend gap penalty of 0.1, and using a Gonnetsimilarity matrix (MacVector™ 10.0.2, MacVector Inc., 2008). The lengthof the sequences compared with respect to identity of sequences willdepend upon the particular sequences. In various embodiments, identityis determined by comparing the sequence of a mature protein from itsN-terminal to its C-terminal. In various embodiments when comparing achimeric human/non-human sequence to a human sequence, the human portionof the chimeric human/non-human sequence (but not the non-human portion)is used in making a comparison for the purpose of ascertaining a levelof identity between a human sequence and a human portion of a chimerichuman/non-human sequence (e.g., comparing a human ectodomain of achimeric human/mouse protein to a human ectodomain of a human protein).

The terms “homology” or “homologous” in reference to sequences, e.g.,nucleotide or amino acid sequences, means two sequences which, uponoptimal alignment and comparison, are identical in at least about 75% ofnucleotides or amino acids, at least about 80% of nucleotides or aminoacids, at least about 90-95% nucleotides or amino acids, e.g., greaterthan 97% nucleotides or amino acids. One skilled in the art wouldunderstand that, for optimal gene targeting, the targeting constructshould contain arms homologous to endogenous DNA sequences (i.e.,“homology arms”); thus, homologous recombination can occur between thetargeting construct and the targeted endogenous sequence.

The term “operably linked” refers to a juxtaposition wherein thecomponents so described are in a relationship permitting them tofunction in their intended manner. As such, a nucleic acid sequenceencoding a protein may be operably linked to regulatory sequences (e.g.,promoter, enhancer, silencer sequence, etc.) so as to retain propertranscriptional regulation. In addition, various portions of thechimeric or humanized protein of the invention may be operably linked toretain proper folding, processing, targeting, expression, and otherfunctional properties of the protein in the cell. Unless statedotherwise, various domains of the chimeric or humanized protein of theinvention are operably linked to each other.

The terms “MHC II complex,” “MHC II protein,” or the like, as usedherein, include the complex between an MHC II α polypeptide and an MHCII β polypeptide. The term “MHC II α polypeptide” or “MHC II βpolypeptide” (or the like), as used herein, includes the MHC I αpolypeptide alone or MHC II β polypeptide alone, respectively.Similarly, the terms “HLA-DR4 complex”, “HLA-DR4 protein,” “H-2Ecomplex,” “H-2E” protein,” or the like, refer to complex between α and βpolypeptides. Typically, the terms “human MHC” and “HLA” are usedinterchangeably.

The term “replacement” in reference to gene replacement refers toplacing exogenous genetic material at an endogenous genetic locus,thereby replacing all or a portion of the endogenous gene with anorthologous or homologous nucleic acid sequence. As demonstrated in theExamples below, nucleic acid sequence of endogenous MHC II locus wasreplaced by a nucleotide sequence comprising sequences encoding portionsof human MHC II α and β polypeptides; specifically, encoding theextracellular portions of the MHC II α and β polypeptides.

“Functional” as used herein, e.g., in reference to a functionalpolypeptide, refers to a polypeptide that retains at least onebiological activity normally associated with the native protein. Forexample, in some embodiments of the invention, a replacement at anendogenous locus (e.g., replacement at an endogenous non-human MHC IIlocus) results in a locus that fails to express a functional endogenouspolypeptide.

Genetically Modified MHC II Animals

In various aspects, the invention generally provides geneticallymodified non-human animals that comprise in their genome a nucleotidesequence encoding a human or humanized MHC II complex; thus, the animalsexpress a human or humanized MHC II complex (e.g., MHC II α and βpolypeptides).

MHC genes are categorized into three classes: class I, class II, andclass III, all of which are encoded either on human chromosome 6 ormouse chromosome 17. A schematic of the relative organization of thehuman and mouse MHC classes is presented in FIGS. 2 and 3, respectively.The majority of MHC genes are polymorphic, in fact they are the mostpolymorphic genes of the mouse and human genomes. MHC polymorphisms arepresumed to be important in providing evolutionary advantage; changes insequence can result in differences in peptide binding that allow forbetter antigen presentation. One exception is the human HLA-DRα chainand its mouse homolog, Eα (i.e., H-2Ea), which are monomorphic.

MHC class II complex comprises two non-covalently associated domains: anα chain and a β chain, also referred herein as an α polypeptide and a βpolypeptide (FIG. 1). The protein spans the plasma membrane; thus itcontains an extracellular domain, a transmembrane domain, and acytoplasmic domain. The extracellular portion of the α chain includes α1and α2 domains, and the extracellular portion of the β chain includes β1and β2 domains. The α1 and β1 domains form a peptide-binding cleft onthe cell surface. Due to the three-dimensional confirmation of thepeptide-binding cleft of the MHC II complex, there is theoretically noupper limit on the length of the bound antigen, but typically peptidespresented by MHC II are between 13 and 17 amino acids in length.

In addition to its interaction with the antigenic peptides, thepeptide-binding cleft of the MHC II molecule interacts with invariantchain (Ii) during the processes of MHC II complex formation and peptideacquisition. The α/β MHC II dimers assemble in the endoplasmic reticulumand associate with Ii chain, which is responsible for control of peptidebinding and targeting of the MHC II into endocytic pathway. In theendosome, Ii undergoes proteolysis, and a small fragment of Ii, ClassII-associated invariant chain peptide (CLIP), remains at thepeptide-binding cleft. In the endosome, under control of HLA-DM (inhumans), CLIP is exchanged for antigenic peptides.

MHC II interacts with T cell co-receptor CD4 at the hydrophobic creviceat the junction between α2 and β2 domains. Wang and Reinherz (2002)Structural Basis of T Cell Recognition of Peptides Bound to MHCMolecules, Molecular Immunology, 38:1039-49. When CD4 and T cellreceptor bind the same MHC II molecule complexed with a peptide, thesensitivity of a T cell to antigen is increased, and it requires100-fold less antigen for activation. See, Janeway's Immunobiology,7^(th) Ed., Murphy et al. eds., Garland Science, 2008, incorporatedherein by reference.

Numerous functions have been proposed for transmembrane and cytoplasmicdomains of MHC II. In the case of cytoplasmic domain, it has been shownto be important for intracellular signaling, trafficking to the plasmamembrane, and ultimately, antigen presentation. For example, it wasshown that T cell hybridomas respond poorly to antigen-presenting cells(APCs) transfected with MHC II β chains truncated at the cytoplasmicdomain, and induction of B cell differentiation is hampered. See, e.g.,Smiley et al. (1996) Truncation of the class II β-chain cytoplasmicdomain influences the level of class II/invariant chain-derived peptidecomplexes, Proc. Natl. Acad. Sci. USA, 93:241-44. Truncation of Class IImolecules seems to impair cAMP production. It has been postulated thatdeletion of the cytoplasmic tail of MHC II affects intracellulartrafficking, thus preventing the complex from coming across relevantantigens in the endocytic pathway. Smiley et al. (supra) demonstratedthat truncation of class II molecules at the cytoplasmic domain reducesthe number of CLIP/class II complexes, postulating that this affects theability of CLIP to effectively regulate antigen presentation.

It has been hypothesized that, since MHC II clustering is important forT cell receptor (TCR) triggering, if MHC II molecules truncated at thecytoplasmic domain were prevented from binding cytoskeleton and thusaggregating, antigen presentation to T cells would be affected.Ostrand-Rosenberg et al. (1991) Abrogation of Tumorigenicity by MHCClass II Antigen Expression Requires the Cytoplasmic Domain of the ClassII Molecule, J. Immunol. 147:2419-22. In fact, it was recently shownthat HLA-DR truncated at the cytoplasmic domain failed to associate withthe cytoskeleton following oligomerization. El Fakhy et al. (2004)Delineation of the HLA-DR Region and the Residues Involved in theAssociation with the Cytoskeleton, J. Biol. Chem. 279:18472-80.Importantly, actin cytoskeleton is a site of localized signaltransduction activity, which can effect antigen presentation. Inaddition to association with cytoskeleton, recent studies have alsoshown that up to 20% of all HLA-DR molecules constitutively reside inthe lipid rafts of APCs, which are microdomains rich in cholesterol andglycosphingolipids, and that such localization is important for antigenpresentation, immune synapse formation, and MHC II-mediated signaling.See, e.g., Dolan et al. (2004) Invariant Chain and the MHC IICytoplasmic Domains Regulate Localization of MHC Class II Molecules toLipid Rafts in Tumor Cell-Based Vaccines, J. Immunol. 172:907-14. Dolanet al. suggested that truncation of cytoplasmic domain of MHC II reducesconstitutive localization of MHC II to lipid rafts.

In addition, the cytoplasmic domain of MHC II, in particular the βchain, contains a leucine residue that is subject to ubiquitination byubiquitin ligase, membrane-associated RING-CH I (MARCH I), whichcontrols endocytic trafficking, internalization, and degradation of MHCII; and it has been shown that MARCH-mediated ubiquitination ceases upondendritic cell maturation resulting in increased levels of MHC II at theplasma membrane. Shin et al. (2006) Surface expression of MHC class IIin dendritic cells is controlled by regulated ubiquitination, Nature444:115-18; De Gassart et al. (2008) MHC class II stabilization at thesurface of human dendritic cells is the result of maturation-dependentMARCH I down-regulation, Proc. Natl. Acad. Sci. USA 105:3491-96.

Transmembrane domains of α and β chains of MHC II interact with eachother and this interaction is important for proper assembly of class IIMHC complex. Cosson and Bonifacino (1992) Role of Transmembrane DomainInteractions in the Assembly of Class II MHC Molecules, Nature258:659-62. In fact, MHC II molecules in which the transmembrane domainsof the α and β chains were replaced by the α chain of IL-2 receptor wereretained in the ER and were barely detectable at the cell surface. Id.Through mutagenesis studies, conserved Gly residues at the α and βtransmembrane domains were found to be responsible for MHC II assemblyat the cell surface. Id. Thus, both transmembrane and cytoplasmicdomains are crucial for the proper function of the MHC II complex.

In various embodiments, the invention provides a genetically modifiednon-human animal (e.g., mouse, rat, rabbit, etc.) that comprises in itsgenome a nucleotide sequence encoding a human or humanized MHC IIcomplex, e.g., a human or humanized MHC II α and/or β polypeptide(s).The non-human animal may comprise in its genome a nucleotide sequencethat encodes an MHC II complex that is partially human and partiallynon-human, e.g., a non-human animal that expresses a chimerichuman/non-human MHC II complex (e.g., a non-human animal that expresseschimeric human/non-human MHC II α and β polypeptides). In one aspect,the non-human animal only expresses the human or humanized MHC IIcomplex, e.g., a chimeric human/non-human MHC II complex, and does notexpress an endogenous non-human MHC II complex from an endogenous MHC IIlocus. In some embodiments, the animal is incapable of expressing anyendogenous non-human MHC II complex from an endogenous MHC II locus, butonly expresses the human or humanized MHC II complex. In variousembodiments, the genetically modified non-human animal (e.g., mouse,rat, rabbit, etc.) comprises in its germline a nucleotide sequenceencoding a human or humanized MHC II complex, e.g., a human or humanizedMHC II α and/or β polypeptide(s).

In one aspect, a chimeric human/non-human MHC II complex is provided. Inone embodiment, the chimeric human/non-human MHC II complex comprises achimeric human/non-human MHC II α polypeptide and a chimerichuman/non-human MHC II β polypeptide. In one aspect, a human portion ofthe chimeric MHC II α polypeptide and/or a human portion of the chimericMHC II β polypeptide comprises a peptide-binding domain of a human MHCII α polypeptide and/or human MHC II β polypeptide, respectively. In oneaspect, a human portion of the chimeric MHC II α and/or β polypeptidecomprises an extracellular domain of a human MHC II α and/or βpolypeptide, respectively. In one embodiment, a human portion of thechimeric MHC II α polypeptide comprises al domain of a human MHC II αpolypeptide; in another embodiment, a human portion of the chimeric MHCII α polypeptide comprises α1 and α2 domains of a human MHC II αpolypeptide. In an additional embodiment, a human portion of thechimeric MHC II β polypeptide comprises β1 domain of a human MHC II βpolypeptide; in another embodiment, a human portion of the chimeric MHCII β polypeptide comprises β1 and β2 domains of a human MHC II βpolypeptide.

The human portion of the MHC II α and β polypeptides described hereinmay be encoded by any of HLA-DP, -DQ, and -DR loci. A list of commonlyused HLA antigens and alleles is described in Shankarkumar et al.((2004) The Human Leukocyte Antigen (HLA) System, Int. J. Hum. Genet.4(2):91-103), incorporated herein by reference. Shankarkumar et al. alsopresent a brief explanation of HLA nomenclature used in the art.Additional information regarding HLA nomenclature and various HLAalleles can be found in Holdsworth et al. (2009) The HLA dictionary2008: a summary of HLA-A, -B, -C, -DRB1/3/4/5, and DQB1 alleles andtheir association with serologically defined HLA-A, -B, -C, -DR, and -DQantigens, Tissue Antigens 73:95-170, and a recent update by Marsh et al.(2010) Nomenclature for factors of the HLA system, 2010, Tissue Antigens75:291-455, both incorporated herein by reference. Thus, the human orhumanized MHC II polypeptide may be derived from any functional humanHLA molecules described therein.

In one specific aspect, the human portions of the humanized MHC IIcomplex described herein are derived from human HLA-DR, e.g., HLA-DR4.Typically, HLA-DR α chains are monomorphic, e.g., the α chain of HLA-DRcomplex is encoded by HLA-DRA gene (e.g., HLA-DRα*01 gene). On the otherhand, the HLA-DR β chain is polymorphic. Thus, HLA-DR4 comprises an αchain encoded by HLA-DRA gene and a β chain encoded by HLA-DRB1 gene(e.g., HLA-DRβ1*04 gene). As described herein below, HLA-DR4 is known tobe associated with incidence of a number of autoimmune diseases, e.g.,rheumatoid arthritis, type I diabetes, multiple sclerosis, etc. In oneembodiment of the invention, the HLA-DRA allele is HLA-DRα*01 allele,e.g., HLA-DRα*01:01:01:01. In another embodiment, the HLA-DRB allele isHLA-DRβ1*04, e.g., HLA-DRβ1*04:01:01. Although the present Examplesdescribe these particular HLA sequences; any suitable HLA-DR sequencesare encompassed herein, e.g., polymorphic variants exhibited in humanpopulation, sequences with one or more conservative or non-conservativeamino acid modifications, nucleic acid sequences differing from thesequences described herein due to the degeneracy of genetic code, etc.

The human portions of the humanized MHC II complex may be encoded bynucleotide sequences of HLA alleles known to be associated with commonhuman diseases. Such HLA alleles include, but are not limited to,HLA-DRB1*0401, -DRB1*0301, -DQA1*0501, -DQB1*0201, -DRB1*1501,-DRB1*1502, -DQB1*0602, -DQA1*0102, -DQA1*0201, -DQB1*0202, -DQA1*0501,and combinations thereof. For a summary of HLA allele/diseaseassociations, see Bakker et al. (2006) A high-resolution HLA and SNPhaplotype map for disease association studies in the extended human MHC,Nature Genetics 38:1166-72 and Supplementary Information, incorporatedherein by reference.

In one aspect, a non-human portion of the chimeric human/non-human MHCII complex comprises transmembrane and/or cytoplasmic domains of anendogenous non-human (e.g., rodent, e.g., mouse, rat, etc.) MHC IIcomplex. Thus, a non-human portion of the chimeric human/non-human MHCII α polypeptide may comprise transmembrane and/or cytoplasmic domainsof an endogenous non-human MHC II α polypeptide. A non-human portion ofthe chimeric human/non-human MHC II β polypeptide may comprisetransmembrane and/or cytoplasmic domains of an endogenous non-human MHCII β polypeptide. In one aspect, the animal is a mouse, and non-humanportions of the chimeric α and β polypeptides are derived from a mouseH-2E protein. Thus, non-human portions of the chimeric α and βpolypeptides may comprise transmembrane and cytoplasmic domains derivedfrom a mouse H-2E protein. Although specific H-2E sequences arecontemplated in the Examples, any suitable sequences, e.g., polymorphicvariants, conservative/non-conservative amino acid substitutions, etc.,are encompassed herein.

In various aspects of the invention, the sequence(s) encoding a chimerichuman/non-human MHC II complex are located at an endogenous non-humanMHC II locus (e.g., mouse H-2A and/or H-2E locus). In one embodiment,this results in a replacement of an endogenous MHC II gene(s) or aportion thereof with a nucleotide sequence(s) encoding a human orhumanized MHC II protein, e.g., a chimeric gene encoding a chimerichuman/non-human MHC II protein described herein. Since the nucleotidesequences encoding MHC II α and β polypeptides are located in proximityto one another on the chromosome, a replacement can be designed totarget the two genes either independently or together; both of thesepossibilities are encompassed herein. In one embodiment, the replacementcomprises a replacement of an endogenous nucleotide sequence encoding anMHC II α and β polypeptides with a nucleotide sequence encoding achimeric human/non-human MHC α polypeptide and a chimerichuman/non-human MHC β polypeptide. In one aspect, the replacementcomprises replacing nucleotide sequences representing one or more (e.g.,two) endogenous MHC II genes. Thus, the non-human animal contains achimeric human/non-human nucleotide sequence at an endogenous MHC IIlocus, and expresses a chimeric human/non-human MHC II protein from theendogenous non-human locus.

Thus, provided herein is a non-human animal comprising at an endogenousMHC II gene locus a first nucleotide sequence encoding a chimerichuman/non-human MHC II α polypeptide and a second nucleotide sequenceencoding a chimeric human/non-human MHC II β polypeptide, wherein ahuman portion of the chimeric human/non-human MHC II α polypeptidecomprises a human MHC II α extracellular domain and a human portion ofthe chimeric human/non-human MHC II β polypeptide comprises a human MHCII β extracellular domain, and wherein the chimeric human/non-human MHCII α and MHC II β polypeptides form a functional MHC II complex on asurface of a cell.

A chimeric human/non-human polypeptide may be such that it comprises ahuman or a non-human leader (signal) sequence. In one embodiment, thechimeric MHC II α polypeptide comprises a non-human leader sequence ofan endogenous MHC II α polypeptide. In one embodiment, the chimeric MHCII β polypeptide comprises a non-human leader sequence of an endogenousMHC II β polypeptide. In an alternative embodiment, the chimeric MHC IIα and/or MHC II β polypeptide comprises a non-human leader sequence ofMHC II α and/or MHC II β polypeptide, respectively, from anothernon-human animal, e.g., another rodent or another mouse strain. Thus,the nucleotide sequence encoding the chimeric MHC II α and/or MHC II βpolypeptide may be operably linked to a nucleotide sequence encoding anon-human MHC II α and/or MHC II β leader sequence, respectively. In yetanother embodiment, the chimeric MHC II α and/or MHC II β polypeptidecomprises a human leader sequence of human MHC II α and/or human MHC IIβ polypeptide, respectively (e.g., a leader sequence of human HLA-DRAand/or human HLA-DRβ1*04, respectively).

A chimeric human/non-human MHC II α and/or MHC II β polypeptide maycomprise in its human portion a complete or substantially completeextracellular domain of a human MHC II α and/or human MHC II βpolypeptide, respectively. Thus, a human portion may comprise at least80%, preferably at least 85%, more preferably at least 90%, e.g., 95% ormore of the amino acids encoding an extracellular domain of a human MHCII α and/or human MHC II β polypeptide (e.g., human HLA-DRA and/or humanHLA-DRβ1*04). In one example, substantially complete extracellulardomain of the human MHC II α and/or human MHC II β polypeptide lacks ahuman leader sequence. In another example, the chimeric human/non-humanMHC II α and/or the chimeric human/non-human MHC II β polypeptidecomprises a human leader sequence.

Moreover, the chimeric MHC II α and/or MHC II β polypeptide may beexpressed under the control of endogenous non-human promoter andregulatory elements, e.g., mouse MHC II α and/or MHC II β regulatoryelements, respectively. Such arrangement will facilitate properexpression of the chimeric MHC II polypeptides in the non-human animal,e.g., during immune response in the non-human animal.

The genetically modified non-human animal may be selected from a groupconsisting of a mouse, rat, rabbit, pig, bovine (e.g., cow, bull,buffalo), deer, sheep, goat, chicken, cat, dog, ferret, primate (e.g.,marmoset, rhesus monkey). For the non-human animals where suitablegenetically modifiable ES cells are not readily available, other methodsare employed to make a non-human animal comprising the geneticmodification. Such methods include, e.g., modifying a non-ES cell genome(e.g., a fibroblast or an induced pluripotent cell) and employingnuclear transfer to transfer the modified genome to a suitable cell,e.g., an oocyte, and gestating the modified cell (e.g., the modifiedoocyte) in a non-human animal under suitable conditions to form anembryo.

In one aspect, the non-human animal is a mammal. In one aspect, thenon-human animal is a small mammal, e.g., of the superfamily Dipodoideaor Muroidea. In one embodiment, the genetically modified animal is arodent. In one embodiment, the rodent is selected from a mouse, a rat,and a hamster. In one embodiment, the rodent is selected from thesuperfamily Muroidea. In one embodiment, the genetically modified animalis from a family selected from Calomyscidae (e.g., mouse-like hamsters),Cricetidae (e.g., hamster, New World rats and mice, voles), Muridae(true mice and rats, gerbils, spiny mice, crested rats), Nesomyidae(climbing mice, rock mice, with-tailed rats, Malagasy rats and mice),Platacanthomyidae (e.g., spiny dormice), and Spalacidae (e.g., molerates, bamboo rats, and zokors). In a specific embodiment, thegenetically modified rodent is selected from a true mouse or rat (familyMuridae), a gerbil, a spiny mouse, and a crested rat. In one embodiment,the genetically modified mouse is from a member of the family Muridae.In one embodiment, the animal is a rodent. In a specific embodiment, therodent is selected from a mouse and a rat. In one embodiment, thenon-human animal is a mouse.

In a specific embodiment, the non-human animal is a rodent that is amouse of a C57BL strain selected from C57BL/A, C57BL/An, C57BL/GrFa,C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10,C57BL/10ScSn, C57BL/10Cr, and C57BL/OIa. In another embodiment, themouse is a 129 strain selected from the group consisting of a strainthat is 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1/SV, 129S1/SvIm),129S2, 129S4, 129S5, 129S9/SvEvH, 129S6 (129/SvEvTac), 129S7, 129S8,129T1, 129T2 (see, e.g., Festing et al. (1999) Revised nomenclature forstrain 129 mice, Mammalian Genome 10:836, see also, Auerbach et al(2000) Establishment and Chimera Analysis of 129/SvEv- andC57BL/6-Derived Mouse Embryonic Stem Cell Lines). In a specificembodiment, the genetically modified mouse is a mix of an aforementioned129 strain and an aforementioned C57BL/6 strain. In another specificembodiment, the mouse is a mix of aforementioned 129 strains, or a mixof aforementioned BL/6 strains. In a specific embodiment, the 129 strainof the mix is a 129S6 (129/SvEvTac) strain. In another embodiment, themouse is a BALB strain, e.g., BALB/c strain. In yet another embodiment,the mouse is a mix of a BALB strain and another aforementioned strain.

In one embodiment, the non-human animal is a rat. In one embodiment, therat is selected from a Wistar rat, an LEA strain, a Sprague Dawleystrain, a Fischer strain, F344, F6, and Dark Agouti. In one embodiment,the rat strain is a mix of two or more strains selected from the groupconsisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and DarkAgouti.

Thus, in one embodiment, the invention relates to a genetically modifiedmouse that comprises in its genome a nucleotide sequence encoding achimeric human/mouse MHC II complex, e.g., chimeric human/mouse MHC II αand β polypeptides. In one embodiment, a human portion of the chimerichuman/mouse MHC II α polypeptide comprises a human MHC II α peptidebinding or extracellular domain and a human portion of the chimerichuman/mouse MHC II β polypeptide comprises a human MHC II β peptidebinding or extracellular domain. In some embodiments, the mouse does notexpress a peptide binding or an extracellular domain of endogenous mouseα and/or β polypeptide from an endogenous mouse locus (e.g., H-2A and/orH-2E locus). In some embodiments, the mouse comprises a genome thatlacks a gene that encodes a functional MHC class II molecule comprisingan H-2Ab1, H-2Aa, H-2Eb1, H-2Eb2, H-2Ea, and a combination thereof. Thepeptide-binding domain of the human MHC II α polypeptide may comprise α1domain and the peptide-binding domain of the human MHC II β polypeptidemay comprise a β1 domain; thus, the peptide-binding domain of thechimeric MHC II complex may comprise human α1 and β1 domains. Theextracellular domain of the human MHC II α polypeptide may comprise α1and α2 domains and the extracellular domain of the human MHC II βpolypeptide may comprise β1 and β2 domains; thus, the extracellulardomain of the chimeric MHC II complex may comprise human α1, α2, β1 andβ2 domains. In one embodiment, the mouse portion of the chimeric MHC IIcomplex comprises transmembrane and cytosolic domains of mouse MHC II,e.g. mouse H-2E (e.g., transmembrane and cytosolic domains of mouse H-2Eα and β chains).

Therefore, in one embodiment, a genetically modified mouse is provided,wherein the mouse comprises at an endogenous mouse MHC II locus a firstnucleotide sequence encoding a chimeric human/mouse MHC II α polypeptideand a second nucleotide sequence encoding a chimeric human/mouse MHC IIβ polypeptide, wherein a human portion of the chimeric MHC II αpolypeptide comprises an extracellular domain derived from an αpolypeptide of a human HLA-DR4 protein and the human portion of thechimeric MHC II β polypeptide comprises an extracellular domain derivedfrom a β polypeptide of a human HLA-DR4 protein, wherein a mouse portionof the chimeric MHC II α polypeptide comprises transmembrane andcytoplasmic domains of a mouse H-2E α chain and a mouse portion of thechimeric MHC II β polypeptide comprises transmembrane and cytoplasmicdomains of a mouse H-2E β chain, and wherein the mouse expresses afunctional chimeric HLA-DR4/H-2E MHC II complex. In one embodiment thechimeric HLA-DR4/H-2E MHC II complex comprises an MHC II α chain thatincludes extracellular domains (e.g., α1, and α2 domains) derived fromHLA-DR4 protein (HLA-DRA α1, and α2 domains) and transmembrane andcytoplasmic domains from a mouse H-2E α chain, as well as an MHC II βchain that includes extracellular domains (e.g., β1 and β2 domains)derived from HLA-DR4 (HLA-DRβ1*04 β1 and β2 domains) and transmembraneand cytoplasmic domains from mouse H-2E β chain. In one aspect, themouse does not express functional endogenous H-2A and H-2E polypeptidesfrom their endogenous mouse loci (e.g., the mouse does not expressH-2Ab1, H-2Aa, H-2Eb1, H-2Eb2, and H-2Ea polypeptides). In variousembodiments, expression of the first and second nucleotide sequences isunder the control of respective endogenous mouse promoters andregulatory elements. In various embodiments of the invention, the firstand the second nucleotide sequences are located on the same chromosome.In some aspects, the mouse comprises two copies of the chimeric MHC IIlocus containing the first and the second nucleotide sequences, while inother aspects, the mouse comprises one copy of the MHC II locuscontaining the first and the second nucleotide sequences. Thus, themouse may be homozygous or heterozygous for the chimeric MHC II locuscontaining the first and the second nucleotide sequences. In variousembodiments, the first and the second nucleotide sequences are comprisesin the germline of the mouse.

In some embodiments described herein, a mouse is provided that comprisesa chimeric MHC II locus at an endogenous mouse MHC II locus, e.g., viareplacement of endogenous mouse H-2A and H-2E genes. In some aspects,the chimeric locus comprises a nucleotide sequence that encodes anextracellular domain of a human HLA-DRA and transmembrane andcytoplasmic domains of a mouse H-2E α chain, as well as an extracellulardomain of a human HLA-DRβ1*04 and transmembrane and cytoplasmic domainsof a mouse H-2E β chain. The various domains of the chimeric locus arelinked in such a fashion that the locus expresses a functional chimerichuman/mouse MHC II complex.

In various embodiments, a non-human animal (e.g., a rodent, e.g., amouse or rat) that expresses a functional chimeric MHC II protein from achimeric MHC II locus as described herein displays the chimeric proteinon a cell surface. In one embodiment, the non-human animal expresses thechimeric MHC II protein on a cell surface in a cellular distributionthat is the same as observed in a human. In one aspect, the celldisplays a peptide fragment (antigen fragment) bound to an extracellularportion (e.g., human HLA-DR4 extracellular portion) of the chimeric MHCII protein.

In various embodiments, a cell displaying the chimeric MHC II protein,e.g., HLA-DR4/H-2E protein, is an antigen-presenting cell (APC) e.g., amacrophage, a dendritic cell, or a B cell. In some embodiments, thepeptide fragment presented by the chimeric protein is derived from atumor. In other embodiments, the peptide fragment presented by thechimeric MHC II protein is derived from a pathogen, e.g., a bacterium, avirus, or a parasite.

The chimeric MHC II protein described herein may interact with otherproteins on the surface of the same cell or a second cell. In someembodiments, the chimeric MHC II protein interacts with endogenousnon-human proteins on the surface of said cell. The chimeric MHC IIprotein may also interact with human or humanized proteins on thesurface of the same cell or a second cell. In some embodiments, thesecond cell is a T cell, and the chimeric MHC II protein interacts withT cell receptor (TCR) and its co-receptor CD4. In some embodiments, theT cell is an endogenous mouse T cell. In other embodiments, the T cellis a human T cell. In some embodiments, the TCR is a human or humanizedTCR. In additional embodiments, the CD4 is a human or humanized CD4. Inother embodiment, either one or both of TCR and CD4 are non-human, e.g.,mouse or rat.

In one embodiment, a genetically modified non-human animal as describedherein is provided that does not develop tumors at a higher rate than awild-type animal that lacks a chimeric MHC II gene. In some embodiments,the animal does not develop hematological malignancies, e.g., various Tand B cell lymphomas, leukemias, composite lymphomas (e.g., Hodgkin'slymphoma), at a higher rate than the wild-type animal.

In addition to a genetically engineered non-human animal, a non-humanembryo (e.g., a rodent, e.g., a mouse or a rat embryo) is also provided,wherein the embryo comprises a donor ES cell that is derived from anon-human animal (e.g., a rodent, e.g., a mouse or a rat) as describedherein. In one aspect, the embryo comprises an ES donor cell thatcomprises the chimeric MHC II gene, and host embryo cells.

Also provided is a tissue, wherein the tissue is derived from anon-human animal (e.g., a rodent, e.g., a mouse or a rat) as describedherein, and expresses the chimeric MHC II protein (e.g., HLA-DR4/H-2Eprotein).

In addition, a non-human cell isolated from a non-human animal asdescribed herein is provided. In one embodiment, the cell is an ES cell.In one embodiment, the cell is an antigen-presenting cell, e.g.,dendritic cell, macrophage, B cell. In one embodiment, the cell is animmune cell. In one embodiment, the immune cell is a lymphocyte.

Also provided is a non-human cell comprising a chromosome or fragmentthereof of a non-human animal as described herein. In one embodiment,the non-human cell comprises a nucleus of a non-human animal asdescribed herein. In one embodiment, the non-human cell comprises thechromosome or fragment thereof as the result of a nuclear transfer.

In one aspect, a non-human induced pluripotent cell comprising geneencoding a chimeric MHC II protein (e.g., HLA-DR4/H-2E protein) asdescribed herein is provided. In one embodiment, the induced pluripotentcell is derived from a non-human animal as described herein.

In one aspect, a hybridoma or quadroma is provided, derived from a cellof a non-human animal as described herein. In one embodiment, thenon-human animal is a mouse or rat.

In one aspect, an in vitro preparation is provided that comprises afirst cell that bears a chimeric human/rodent MHC II surface proteinthat comprises a bound peptide to form a chimeric human/rodent MHCII/peptide complex, and a second cell that binds the chimerichuman/rodent MHC II/peptide complex. In one embodiment, the second cellcomprises a human or humanized T-cell receptor, and in one embodimentfurther comprises a human or humanized CD4. In one embodiment, thesecond cell is a rodent (e.g., mouse or rat) cell comprising a human orhumanized T-cell receptor and a human or humanized CD4 protein. In oneembodiment, the second cell is a human cell.

Also provided is a method for making a genetically engineered non-humananimal (e.g., a genetically engineered rodent, e.g., a mouse or rat)described herein. The method for making a genetically engineerednon-human animal results in the animal whose genome comprises anucleotide sequence encoding a chimeric MHC II protein (e.g., chimericMHC II α and β polypeptides). In one embodiment, the method results in agenetically engineered mouse, whose genome comprises at an endogenousMHC II locus a nucleotide sequence encoding a chimeric human/mouse MHCII protein, wherein a human portion of the chimeric MHC II proteincomprises an extracellular domain of a human HLA-DR4 and a mouse portioncomprises transmembrane and cytoplasmic domains of a mouse H-2E. In someembodiments, the method utilizes a targeting construct made usingVELOCIGENE® technology, introducing the construct into ES cells, andintroducing targeted ES cell clones into a mouse embryo usingVELOCIMOUSE® technology, as described in the Examples. In oneembodiment, the ES cells are a mix of 129 and C57BL/6 mouse strains; inone embodiment, the ES cells are a mix of BALB/c and 129 mouse strains.

A nucleotide construct used for generating genetically engineerednon-human animals described herein is also provided. In one aspect, thenucleotide construct comprises: 5′ and 3′ non-human homology arms, a DNAfragment comprising human HLA-DR α and β chain sequences, and aselection cassette flanked by recombination sites. In one embodiment,the human HLA-DR α and β chain sequences are genomic sequences thatcomprise introns and exons of human HLA-DR α and β chain genes. In oneembodiment, the non-human homology arms are homologous to non-human MHCII genomic sequence.

In one embodiment, the human HLA-DR α chain sequence comprises an α1 andα2 domain coding sequence. In a specific embodiment, it comprises, from5′ to 3′: al exon (exon 2), α1/α2 intron (intron 2), and α2 exon (exon3). In one embodiment, the human HLA-DR β chain sequence comprises a β1and β2 domain coding sequence. In a specific embodiment, it comprises,from 5′ to 3′: β1 exon (exon 2), β1/β2 intron (intron 2), and β2 exon(exon 3).

A selection cassette is a nucleotide sequence inserted into a targetingconstruct to facilitate selection of cells (e.g., ES cells) that haveintegrated the construct of interest. A number of suitable selectioncassettes are known in the art. Commonly, a selection cassette enablespositive selection in the presence of a particular antibiotic (e.g.,Neo, Hyg, Pur, CM, SPEC, etc.). In addition, a selection cassette may beflanked by recombination sites, which allow deletion of the selectioncassette upon treatment with recombinase enzymes. Commonly usedrecombination sites are loxP and Frt, recognized by Cre and Flp enzymes,respectively, but others are known in the art. A selection cassette maybe located anywhere in the construct outside the coding region. In oneembodiment, the selection cassette is located in the β chain intron,e.g., β2/transmembrane domain intron (intron 3).

In one embodiment, 5′ and 3′ homology arms comprise genomic sequence at5′ and 3′ locations of endogenous non-human MHC II locus. In oneembodiment, the 5′ homology arm comprises genomic sequence upstream ofmouse H-2Ab1 gene and the 3′ homology arm comprises genomic sequencedownstream of mouse H-2Ea gene. In this embodiment, the construct allowsreplacement of both mouse H-2E and H-2A genes.

Thus, in one aspect, a nucleotide construct is provided comprising, from5′ to 3′: a 5′ homology arm containing mouse genomic sequence upstreamof mouse H-2Ab1 gene, a first nucleotide sequence comprising a sequenceencoding a chimeric human/mouse MHC II β chain, a second nucleotidesequence comprising a sequence encoding a chimeric human/mouse MHC II αchain, and a 3′ homology arm containing mouse genomic sequencedownstream of mouse H-2Ea gene. In a specific embodiment, the firstnucleotide sequence comprising a sequence encoding a chimerichuman/mouse MHC II β chain comprises human β1 exon, β1/β2 intron, β2exon, an a selection cassette flanked by recombination sites inserted inthe intronic region between the human β2 exon sequence and the sequenceof a mouse transmembrane domain exon. In a specific embodiment, thesecond nucleotide sequence comprising a sequence encoding a chimerichuman/mouse MHC II α chain comprises human α1 exon, α1/α2 intron, andhuman α2 exon. An exemplary construct of the invention is depicted inFIG. 5 (MAID 1680).

Upon completion of gene targeting, ES cells or genetically modifiednon-human animals are screened to confirm successful incorporation ofexogenous nucleotide sequence of interest or expression of exogenouspolypeptide. Numerous techniques are known to those skilled in the art,and include (but are not limited to) Southern blotting, long PCR,quantitative PCT (e.g., real-time PCR using TAQMAN®), fluorescence insitu hybridization, Northern blotting, flow cytometry, Western analysis,immunocytochemistry, immunohistochemistry, etc. In one example,non-human animals (e.g., mice) bearing the genetic modification ofinterest can be identified by screening for loss of mouse allele and/orgain of human allele using a modification of allele assay described inValenzuela et al. (2003) High-throughput engineering of the mouse genomecoupled with high-resolution expression analysis, Nature Biotech.21(6):652-659. Other assays that identify a specific nucleotide or aminoacid sequence in the genetically modified animals are known to thoseskilled in the art.

The disclosure also provides a method of modifying an MHC II locus of anon-human animal to express a chimeric human/non-human MHC II complexdescribed herein. In one embodiment, the invention provides a method ofmodifying an MHC II locus of a mouse to express a chimeric human/mouseMHC II complex comprising replacing at the endogenous mouse MHC II locusa nucleotide sequence encoding a mouse MHC II complex with a nucleotidesequence encoding a chimeric human/mouse MHC II complex. In a specificaspect, the nucleotide sequence encoding the chimeric human/mouse MHC IIcomplex comprises a first nucleotide sequences encoding an extracellulardomain of a human MHC II α chain (e.g., HLA-DR4 α chain) andtransmembrane and cytoplasmic domains of a mouse MHC II α chain (e.g.,H-2E α chain) and a second nucleotide sequence encoding an extracellulardomain of a human MHC II β chain (e.g., HLA-DR4 β chain) andtransmembrane and cytoplasmic domains of a mouse MHC II β chain (e.g.,H-2E β chain, e.g., H-2Eb1 chain). In some embodiments, the modifiedmouse MHC II locus expresses a chimeric HLA-DR4/H-2E protein.

In one aspect, a method for making a chimeric human HLA classII/non-human MHC class II molecule is provided, comprising expressing ina single cell a chimeric HLA-DR4/H-2E protein from a nucleotideconstruct as described herein. In one embodiment, the nucleotideconstruct is a viral vector; in a specific embodiment, the viral vectoris a lentiviral vector. In one embodiment, the cell is selected from aCHO, COS, 293, HeLa, and a retinal cell expressing a viral nucleic acidsequence (e.g., a PERC.6™ cell).

In one aspect, a cell that expresses a chimeric HLA-DR4/H-2E protein isprovided. In one embodiment, the cell comprises an expression vectorcomprising a chimeric MHC class II sequence as described herein. In oneembodiment, the cell is selected from CHO, COS, 293, HeLa, and a retinalcell expressing a viral nucleic acid sequence (e.g., a PERC.6™ cell).

A chimeric MHC class II molecule made by a non-human animal as describedherein is also provided, wherein the chimeric MHC class II moleculecomprises α1, α2, β1, and β2 domains from a human MHC II protein, e.g.,HLA-DR4 protein, and transmembrane and cytoplasmic domains from anon-human MHC II protein, e.g., mouse H-2E protein. The chimeric MHC IIcomplex comprising an extracellular domain of HLA-DR4 described hereinmaybe detected by anti-HLA-DR antibodies. Thus, a cell displayingchimeric human/non-human MHC II polypeptide may be detected and/orselected using anti-HLA-DR antibody.

Although the Examples that follow describe a genetically engineeredanimal whose genome comprises a replacement of a nucleotide sequenceencoding mouse H-2A and H-2E proteins with a nucleotide sequenceencoding a chimeric human/mouse HLA-DR4/H-2E protein, one skilled in theart would understand that a similar strategy may be used to introducechimeras comprising other human MHC II genes (HLA-DP and HLA-DQ). Thus,an additional embodiment of the invention is directed to a geneticallyengineered animal whose genome comprises a nucleotide sequence encodinga chimeric HLA-DQ/H-2A protein. In one embodiment, the nucleotidesequence encodes a chimeric HLA-DQ2.5/H-2A protein. In anotherembodiment, the nucleotide sequence encodes a chimeric HLA-DQ8/H-2Aprotein. In addition, introduction of multiple humanized MHC IImolecules (e.g., chimeric HLA-DR/H-2E and HLA-DQ/H-2A) is alsocontemplated.

Use of Genetically Modified Animals

In various embodiments, the genetically modified non-human animalsdescribed herein make APCs with human or humanized MHC II on the cellsurface and, as a result, present peptides derived from cytosolicproteins as epitopes for T cells in a human-like manner, becausesubstantially all of the components of the complex are human orhumanized. The genetically modified non-human animals of the inventioncan be used to study the function of a human immune system in thehumanized animal; for identification of antigens and antigen epitopesthat elicit immune response (e.g., T cell epitopes, e.g., unique humancancer epitopes), e.g., for use in vaccine development; for evaluationof vaccine candidates and other vaccine strategies; for studying humanautoimmunity; for studying human infectious diseases; and otherwise fordevising better therapeutic strategies based on human MHC expression.

MHC II complex binds peptides derived from extracellular proteins, e.g.,extracellular bacterium, neighboring cells, or polypeptides bound by Bcell receptors and internalized into a B cell. Once extracellularproteins enter endocytic pathway, they are degraded into peptides, andpeptides are bound and presented by MHC II. Once a peptide presented byMHC II is recognized by CD4+ T cells, T cells are activated,proliferate, differentiate to various T helper subtypes (e.g., T_(H)1,T_(H)2), and lead to a number of events including activation ofmacrophage-mediated pathogen killing, B cell proliferation, and antibodyproduction. Because of MHC II role in immune response, understanding ofMHC II peptide presentation is important in the development of treatmentfor human pathologies. However, presentation of antigens in the contextof mouse MHC II is only somewhat relevant to human disease, since humanand mouse MHC complexes recognize antigens differently, e.g., a mouseMHC II may not recognize the same antigens or may present differentepitopes than a human MHC II. Thus, the most relevant data for humanpathologies is obtained through studying the presentation of antigenepitopes by human MHC II.

Thus, in various embodiments, the genetically engineered animals of thepresent invention are useful, among other things, for evaluating thecapacity of an antigen to initiate an immune response in a human, andfor generating a diversity of antigens and identifying a specificantigen that may be used in human vaccine development.

In one aspect, a method for determining antigenicity in a human of apeptide sequence is provided, comprising exposing a genetically modifiednon-human animal as described herein to a molecule comprising thepeptide sequence, allowing the non-human animal to mount an immuneresponse, and detecting in the non-human animal a cell that binds asequence of the peptide presented by a humanized MHC II complexdescribed herein.

In one aspect, a method for determining whether a peptide will provokean immune response in a human is provided, comprising exposing agenetically modified non-human animal as described herein to thepeptide, allowing the non-human animal to mount an immune response, anddetecting in the non-human animal a cell that binds a sequence of thepeptide by a chimeric human/non-human MHC class II molecule as describedherein. In one embodiment, the non-human animal following exposurecomprises an MHC class II-restricted CD4+ T cell that binds the peptide.

In one aspect, a method for identifying a human CD4+ T cell epitope isprovided, comprising exposing a non-human animal as described herein toan antigen comprising a putative T cell epitope, allowing the non-humananimal to mount an immune response, and identifying the epitope bound bythe MHC class II-restricted CD4+ T cell.

In one aspect, a method is provided for identifying an antigen thatgenerates a CD4+ T cell response in a human, comprising exposing aputative antigen to a mouse as described herein, allowing the mouse togenerate an immune response, detecting a CD4+ T cell response that isspecific for the antigen in the context of a human MHC II molecule(e.g., an HLA-DR molecule), and identifying the antigen bound by thehuman MHC II-restricted molecule (e.g., human HLA-DR restrictedmolecule).

In one embodiment, the antigen comprises a bacterial protein. In oneembodiment, the antigen comprises a human tumor cell antigen. In oneembodiment, the antigen comprises a putative vaccine for use in a human,or another biopharmaceutical. In one embodiment, the antigen comprises ahuman epitope that generates antibodies in a human. In yet anotherembodiment, an antigen comprises a yeast or fungal cell antigen. In yetanother embodiment, an antigen is derived from a human parasite.

In one aspect, a method is provided for determining whether a putativeantigen contains an epitope that upon exposure to a human immune systemwill generate an HLA-DR-restricted immune response (e.g.,HLA-DR4-restricted response), comprising exposing a mouse as describedherein to the putative antigen and measuring an antigen-specificHLA-DR-restricted (e.g., HLA-DR4-restricted) immune response in themouse. In another aspect, a method is provided for determining wherein aputative antigen contains an epitope that upon exposure to a humanimmune system will generate an HLA-DQ-restricted response.

Also provided is a method of generating antibodies to an antigen, e.g.,an antigen derived from bacterium, parasite, etc., presented in thecontext of a human MHC II complex, comprising exposing a mouse describedherein to an antigen, allowing a mouse to mount an immune response,wherein the immune response comprises antibody production, and isolatingan antibody that recognizes the antigen presented in the context ofhuman MHC II complex. In one embodiment, in order to generate antibodiesto the peptide-MHC II, the MHC II humanized mouse is immunized with apeptide-MHC II immunogen.

In one aspect, a method for identifying a T cell receptor variabledomain that recognizes an antigen presented in the context of MHC II(e.g., human tumor antigen, a vaccine, etc.) is provided, comprisingexposing a mouse comprising a humanized MHC II complex described hereinto the antigen, allowing the mouse to generate an immune response, andisolating from the mouse a nucleic acid sequence encoding a variabledomain of a T cell receptor that binds MHC II-restricted antigen. In oneembodiment, the antigen is presented in the context of a humanized MHCII (e.g., human HLA II ectodomain/mouse MHC II transmembrane and/orcytoplasmic domain).

The consequence of interaction between a T cell and an APC displaying apeptide in the context of MHC II (e.g., human HLA II ectodomain/mouseMHC II transmembrane and/or cytoplasmic domain) can be measured by anumber of techniques known in the art, e.g., T cell proliferationassays, cytokine release assays, etc.

In addition to the ability to identify antigens and their T cellepitopes from pathogens or neoplasms, the genetically modified animalsof the invention can be used to identify autoantigens of relevance tohuman autoimmune disease, and otherwise study human autoimmune diseaseprogression. It is known that polymorphisms within the HLA loci play arole in predisposition to human autoimmune disease. In fact, specificpolymorphisms in HLA-DR and HLA-DQ loci have been identified thatcorrelate with development of rheumatoid arthritis, type I diabetes,Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, Graves'disease, systemic lupus erythematosus, celiac disease, Crohn's disease,ulcerative colitis, and other autoimmune disorders. See, e.g., Wong andWen (2004) What can the HLA transgenic mouse tell us about autoimmunediabetes?, Diabetologia 47:1476-87; Taneja and David (1998) HLATransgenic Mice as Humanized Mouse Models of Disease and Immunity, J.Clin. Invest. 101:921-26; Bakker et al. (2006), supra; and InternationalMHC and Autoimmunity Genetics Network (2009) Mapping of multiplesusceptibility variants within the MHC region for 7 immune-mediateddiseases, Proc. Natl. Acad. Sci. USA 106:18680-85.

Thus, the methods of making a humanized MHC II complex animals describedherein can be used to introduce MHC II molecules thought to beassociated with specific human autoimmune diseases, and progression ofhuman autoimmune disease can be studied. In addition, non-human animalsdescribed herein can be used to develop animal models of humanautoimmune disease. Mice according to the invention carrying humanizedMHC II proteins described herein can be used to identify potentialautoantigens, to map epitopes involved in disease progression, and todesign strategies for autoimmune disease modulation.

In addition, the genetically modified animals described herein may beused in the study of human allergic response. As allergic responsesappear to be associated with MHC II alleles, genetically modifiedanimals described herein may be used to determine HLA restriction ofallergen specific T cell response and to develop strategies to combatallergic response.

EXAMPLES

The invention will be further illustrated by the following nonlimitingexamples. These Examples are set forth to aid in the understanding ofthe invention but are not intended to, and should not be construed to,limit its scope in any way. The Examples do not include detaileddescriptions of conventional methods that would be well known to thoseof ordinary skill in the art (molecular cloning techniques, etc.).Unless indicated otherwise, parts are parts by weight, molecular weightis average molecular weight, temperature is indicated in Celsius, andpressure is at or near atmospheric.

Example 1. Deletion of the Endogenous MHC Class II H-2A and H-2E Loci

The targeting vector for introducing a deletion of the endogenous MHCclass II H-2Ab1, H-2Aa, H-2Eb1, H-2Eb2, and H-2Ea genes was made usingVELOCIGENE® genetic engineering technology (see, e.g., U.S. Pat. No.6,586,251 and Valenzuela et al., supra). Bacterial Artificial Chromosome(BAC) RP23-458i22 (Invitrogen) DNA was modified to delete the endogenousMHC class II genes H-2Ab1, H-2Aa, H-2Eb1, H-2Eb2, and H-2Ea.

Briefly, upstream and downstream homology arms were derived by PCR ofmouse BAC DNA from locations 5′ of the H-2Ab1 gene and 3′ of the H-2Eagene, respectively. As depicted in FIG. 5, these homology arms were usedto make a cassette that deleted ˜79 kb of RP23-458i22 comprising genesH-2Ab1, H-2Aa, H-2Eb1, H-2Eb2, and H-2Ea of the MHC class II locus bybacterial homologous recombination (BHR). This region was replaced witha hygromycin cassette flanked by lox66 and lox71 sites. The finaltargeting vector from 5′ to 3′ included a 34 kb homology arm comprisingmouse genomic sequence 5′ to the H-2Ab1 gene of the endogenous MHC classII locus, a 5′ lox66 site, a hygromycin cassette, a 3′ lox71 site and a63 kb homology arm comprising mouse genomic sequence 3′ to the H-2Eagene of the endogenous MHC class II locus (MAID 5111, see FIG. 5).

The BAC DNA targeting vector (described above) was used to electroporatemouse ES cells to create modified ES cells comprising a deletion of theendogenous MHC class II locus. Positive ES cells containing a deletedendogenous MHC class II locus were identified by the quantitative PCRassay using TAQMAN™ probes (Lie and Petropoulos (1998) Curr. Opin.Biotechnology 9:43-48). The upstream region of the deleted locus wasconfirmed by PCR using primers 5111U F (CAGAACGCCAGGCTGTAAC; SEQ IDNO:1) and 5111U R (GGAGAGCAGGGTCAGTCAAC; SEQ ID NO:2) and probe 5111U P(CACCGCCACTCACAGCTCCTTACA; SEQ ID NO:3), whereas the downstream regionof the deleted locus was confirmed using primers 5111 D F(GTGGGCACCATCTTCATCATTC; SEQ ID NO:4) and 5111 D R(CTTCCTTTCCAGGGTGTGACTC; SEQ ID NO:5) and probe 5111 D P(AGGCCTGCGATCAGGTGGCACCT; SEQ ID NO:6). The presence of the hygromycincassette from the targeting vector was confirmed using primers HYGF(TGCGGCCGATCTTAGCC; SEQ ID NO:7) and HYGR (TTGACCGATTCCTTGCGG; SEQ IDNO:8) and probe HYGP (ACGAGCGGGTTCGGCCCATTC; SEQ ID NO:9). Thenucleotide sequence across the upstream deletion point (SEQ ID NO:10)included the following, which indicates endogenous mouse sequenceupstream of the deletion point (contained within the parentheses below)linked contiguously to cassette sequence present at the deletion point:(TTTGTAAACA AAGTCTACCC AGAGACAGAT GACAGACTTC AGCTCCAATG CTGATTGGTTCCTCACTTGG GACCAACCCT) CTCGAGTACC GTTCGTATAA TGTATGCTAT ACGAAGTTATATGCATCCGG GTAGGGGAGG. The nucleotide sequence across the downstreamdeletion point (SEQ ID NO:11) included the following, which indicatescassette sequence contiguous with endogenous mouse sequence downstreamof the deletion point (contained within the parentheses below):CCTCGACCTG CAGCCCTAGG ATAACTTCGT ATAATGTATG CTATACGAAC GGTAGAGCTC(CACAGGCATT TGGGTGGGCA GGGATGGACG GTGACTGGGA CAATCGGGAT GGAAGAGCATAGAATGGGAG TTAGGGAAGA). Positive ES cell clones were then used toimplant female mice using the VELOCIMOUSE® method (described below) togenerate a litter of pups containing a deletion of the endogenous MHCclass II locus.

Targeted ES cells described above were used as donor ES cells andintroduced into an 8-cell stage mouse embryo by the VELOCIMOUSE® method(see, e.g., U.S. Pat. No. 7,294,754 and Poueymirou et al. (2007) FOgeneration mice that are essentially fully derived from the donorgene-targeted ES cells allowing immediate phenotypic analyses, NatureBiotech. 25(1):91-99). Mice bearing a deletion of H-2Ab1, H-2Aa, H-2Eb1,H-2Eb2, and H-2Ea genes in the endogenous MHC class II locus wereidentified by genotyping using a modification of allele assay(Valenzuela et al., supra) that detected the presence of the hygromycincassette and confirmed the absence of endogenous MHC class II sequences.

Mice bearing a deletion of H-2Ab1, H-2Aa, H-2Eb1, H-2Eb2, and H-2Eagenes in the endogenous MHC class II locus can be bred to a Cre deletormouse strain (see, e.g., International Patent Application PublicationNo. WO 2009/114400) in order to remove any loxed hygromycin cassetteintroduced by the targeting vector that is not removed, e.g., at the EScell stage or in the embryo. Optionally, the hygromycin cassette isretained in the mice.

Example 2. Generation of Large Targeting Vector (LTVEC) ComprisingHumanized H-2Eb1 and H-2Ea Genes

A targeting vector to introduce humanized MHC II sequences was designedas depicted in FIG. 4. Using VELOCIGENE® genetic engineering technology,Bacterial Artificial Chromosome (BAC) RP23-458i22 DNA was modified invarious steps to: (1) create a vector comprising a functional I-E α exon1 from BALB/c H-2Ea gene (FIG. 4A); (2) create a vector comprisingreplacement of exons 2 and 3 of mouse I-E β gene with those of humanDRβ1*04 and replacement of exons 2 and 3 of mouse I-E α with those ofhuman DRα1*01 (FIG. 4B); (3) create a vector carrying exons 2 and 3 ofhuman DRβ1*04 amongst remaining mouse I-E β exons, and exons 2 and 3 ofhuman DRα1*01 amongst remaining mouse I-E α exons including a functionalI-E α exon 1 from BALB/c mouse (step (1) (FIG. 4C); and (4) remove acryptic splice site in the vector generated in (3) (FIG. 4D).

Specifically, because in the C57Bl/6 mice, the I-E α gene is apseudogene due to the presence of a non-functional exon 1, first, avector comprising a functional I-E α exon 1 from BALB/c H-2Ea gene wascreated (FIG. 4A). RP23-458i22 BAC was modified by bacterial homologousrecombination (1.BHR) to replace chloramphenicol resistance gene withthat of spectromycin. The resultant vector was further modified by BHRto replace the entire I-A and I-E coding region with a neomycin cassetteflanked by recombination sites (2.BHR). Another round of BHR (3. BHR)with the construct comprising an exon encoding BALB/c I-Eα leader(exon 1) and chloramphenicol gene flanked by PI-SceI and I-CeuIrestriction sites resulted in a vector comprising a functional BALB/cH-2Ea exon 1.

Independently, in order to generate a vector comprising replacement ofexons 2 and 3 of mouse I-E β gene with those of human DRβ1*04 andreplacement of exons 2 and 3 of mouse I-E α with those of human DRα1*01,RP23-458i22 BAC was modified via several homologous recombination steps,4. BHR-8. BHR (FIG. 4B). The resultant nucleic acid sequence was flankedby PI-SceI/I-CeuI restriction sites to allow ligation into the constructcarrying BALB/c I-Eα exon 1, mentioned above (FIG. 4C).

The sequence of the final construct depicted in FIG. 4C contained acryptic splice site at the 3′ end of the BALB/c intron. Several BHRsteps (11. BHR-12. BHR) followed by a deletion step were performed toobtain the final targeting vector (MAID 1680) that was used toelectroporate into ES cells (FIG. 4D).

In detail, the final targeting vector (MAID 1680), from 5′ to 3′, wascomprised of a 5′ mouse homology arm consisting of ˜26 kb of mousegenomic sequence ending just upstream of the H-2Ab1 gene of theendogenous MHC class II locus; an ˜59 kb insert containing the humanizedMHC II β chain gene (humanized H-2Eb1 gene) and humanized MHC II α chaingene (humanized H-2Ea gene) and a floxed neomycin cassette; and a 3′mouse homology arm consisting of ˜57 kb of mouse genomic sequencebeginning just downstream of the H-2Ea gene of the endogenous MHC classII locus. The nucleotide sequence across the junction between the 5′ armand the insert (SEQ ID NO:12) included the following: (TGCTGATTGGTTCCTCACTT GGGACCAACC C) TAAGCTTTA TCTATGTCGG GTGCGGAGAA AGAGGTAATGAAATGGCACA AGGAGATCAC ACACCCAAAC CAAACTCGCC, where the italicizedsequence is a unique PI-SceI site, and mouse genomic sequence in the 5′homology arm is in parentheses. The nucleotide sequence across thejunction between the insert and the 3′ arm (SEQ ID NO:13) included thefollowing: CACATCAGTG AGGCTAGAAT AAATTAAAAT CGCTAATATG AAAATGGGG(ATTTGTACCT CTGAGTGTGA AGGCTGGGAA GACTGCTTTC AAGGGAC), where the mousegenomic sequence in the 3′ homology arm is in parentheses.

Within the ˜59 kb insert, the H-2Eb1 gene was modified as follows: a5136 bp region of H-2Eb1, including the last 153 bp of intron1, exon 2,intron 2, exon 3, and the first 122 bp of intron 3, was replaced withthe 3111 bp homologous region of human HLA-DRB1*04, including the last148 bp of intron 1, exon 2, intron 2, exon 3, and the first 132 bp ofintron 3. At the junction between the human and mouse sequences ofintron 3, a cassette consisting of a 5′ lox2372 site, UbC promoter,neomycin resistance gene, and a 3′ lox2372 site, was inserted. Theresulting gene encoded a chimeric HLA-DRB1*04/H-2Eb1 protein comprisedof the mouse H-2Eb1 leader, the human β1 and β2 domains from DRB1*04,and the mouse transmembrane domain and cytoplasmic tail. The nucleotidesequence across the mouse/human junction in intron 1 (SEQ ID NO:14)included the following: (TCCATCACTT CACTGGGTAG CACAGCTGTA ACTGTCCAGCCTG) GGTACCGAGC TCGGATCCAC TAGTAACGGC CGCCAGTGTG CTGGAATTC GCCCTTGATCGAGCTCCCTG GGCTGCAGGT GGTGGGCGTT GCGGGTGGGG CCGGTTAA, where theitalicized sequence is a multiple cloning site introduced during thecloning steps, and the mouse intron 1 sequences are in parentheses. Thenucleotide sequence across the junction between the human intron 3 andneomycin cassette (SEQ ID NO:15) included the following: (ATCTCCATCAGAAGGGCACC GGT) ATAACTT CGTATAAGGT ATCCTATACG AAGTTATATG CATGGCCTCCGCGCCGGGTT, where the 5′ lox2372 site is italicized, and human intron 3sequence is in parentheses. The nucleotide sequence across the junctionbetween the neomycin cassette and mouse intron 3 (SEQ ID NO:16) includedthe following: ATAACTTCGT ATAAGGTATC CTATACGAAG TTATCTCGAG (TGGCTTACAGGTAGGTGCGT GAAGCTTCTA CAAGCACAGT TGCCCCCTGG), where the 3′ lox2372 siteis italicized, and the mouse intron 3 sequence is in parentheses.

Also within the ˜59 kb insert, the H-2Ea gene was modified as follows: a1185 bp region of H-2Ea, including the last 101 bp of intron1, exon 2,intron 2, exon 3, and the first 66 bp of intron 3, was replaced with the1189 bp homologous region of human HLA-DRA1*01, including the last 104bp of intron 1, exon 2, intron 2, exon 3, and the first 66 bp of intron3. As described above, because exon 1 of the C57BL/6 allele of H-2Eacontains a deletion which renders the gene nonfunctional, H-2Ea exon 1and the remainder of intron 1 were replaced with the equivalent 2616 bpregion from the BALB/c allele of H-2Ea, which is functional. Theresulting gene encoded a chimeric H-2Eα/HLA-DRA1*01 protein comprised ofthe mouse H-2Ea leader from BALB/c, the human α1 and α2 domains fromDRA1*01, and the mouse transmembrane domain and cytoplasmic tail. Thenucleotide sequence across the mouse/human junction in intron 1 (SEQ IDNO:17) included the following: (CTGTTTCTTC CCTAACTCCC ATTCTATGCTCTTCCATCCC GA) CCGCGGCCCA ATCTCTCTCC ACTACTTCCT GCCTACATGT ATGTAGGT,where the italicized sequence is a restriction enzyme site introducedduring the cloning steps, and the BALB/c intron 1 sequences are inparentheses. The nucleotide sequence across the human/mouse junction inintron 3 (SEQ ID NO:18) included the following: CAAGGTTTCC TCCTATGATGCTTGTGTGAA ACTCGGGGCC GGCC (AGCATTTAAC AGTACAGGGA TGGGAGCACA GCTCAC),where the italicized sequence is a restriction enzyme site introducedduring the cloning steps, and the mouse intron 3 sequences are inparentheses. The nucleotide sequence across the C57BL/6-BALB/c junction5′ of exon 1 (SEQ ID NO:19) included the following: (GAAAGCAGTCTTCCCAGCCT TCACACTCAG AGGTACAAAT) CCCCATTTTC ATATTAGCGA TTTTAATTTATTCTAGCCTC, where the C57BL/6-specific sequences are in parentheses. Thenucleotide sequence across the BALB/c-C57BL/6 junction 3′ of exon 1 (SEQID NO:20) included the following: TCTTCCCTAA CTCCCATTCT ATGCTCTTCCATCCCGA CCG CGG (CCCAATC TCTCTCCACT ACTTCCTGCC TACATGTATG), where SacIIrestriction site is italicized, and C57BL/6 sequences are inparenthesis.

Example 3. Generation of Humanized MHC II Mice

Simplified diagrams of the strategy for generating humanized MHC II miceusing the vector of Example 2 are presented in FIGS. 5 and 8.

Specifically, MAID1680 BAC DNA (described above) was used toelectroporate MAID5111 ES cells to create modified ES cells comprising areplacement of the endogenous mouse I-A and I-E loci with a genomicfragment comprising a chimeric human DR4/mouse I-E locus. Positive EScells containing deleted endogenous I-A and I-E loci replaced by agenomic fragment comprising a chimeric human DR4/mouse I-E locus wereidentified by a quantitative PCR assay using TAQMAN™ probes (Lie andPetropoulos, supra). The insertion of the human DRα sequences wasconfirmed by PCR using primers hDRA1F (CTGGCGGCTTGAAGAATTTGG; SEQ IDNO:21), hDRA1R (CATGATTTCCAGGTTGGCTTTGTC; SEQ ID NO:22), and probehDRA1P (CGATTTGCCAGCTTTGAGGCTCAAGG; SEQ ID NO:23). The insertion of thehuman DR6 sequences was confirmed by PCR using primers hDRB1F(AGGCTTGGGTGCTCCACTTG; SEQ ID NO:24), hDRB1R (GACCCTGGTGATGCTGGAAAC; SEQID NO:25), and probe hDRB1P (CAGGTGTAAACCTCTCCACTCCGAGGA; SEQ ID NO:26).The loss of the hygromycin cassette from the targeting vector wasconfirmed with primers HYGF (TGCGGCCGATCTTAGCC; SEQ ID NO:7) and HYGR(TTGACCGATTCCTTGCGG; SEQ ID NO:8) and probe HYGP (ACGAGCGGGTTCGGCCCATTC;SEQ ID NO:9).

Positive ES cell clones were then used to implant female mice using theVELOCIMOUSE® method (supra) to generate a litter of pups containing areplacement of the endogenous I-A and I-E loci with a chimeric humanDR4/mouse I-E locus. Targeted ES cells described above were used asdonor ES cells and introduced into an 8-cell stage mouse embryo by theVELOCIMOUSE® method. Mice bearing a chimeric human DR4/mouse I-E locuswere identified by genotyping using a modification of allele assay(Valenzuela et al., supra) that detected the presence of a chimerichuman DR4/mouse I-E locus.

Mice bearing a chimeric human DR4/mouse I-E locus can be bred to a Credeletor mouse strain (see, e.g., International Patent ApplicationPublication No. WO 2009/114400) in order to remove any loxed neomycincassette introduced by the targeting vector that is not removed, e.g.,at the ES cell stage or in the embryo (See FIG. 6).

Example 4. Expression of the Chimeric HLA-DR4 in Genetically ModifiedMice

Spleens from WT or heterozygous humanized HLA-DR4 mice (“1681 HET”) wereperfused with Collagenase D (Roche Bioscience) and erythrocytes werelysed with ACK lysis buffer. Splenocytes were cultured for two days with25 micrograms/mL poly(I:C) to stimulate the expression of MHC-II genes.Cell surface expression of human HLA-DR4 was analyzed by FACS usingfluorochrome-conjugated anti-CD3 (17A2), anti-CD19 (1 D3), anti-CD11c(N418), anti-F480 (BM8), anti-I-A/I-E (M15) and anti-HLADR (L243). Flowcytometry was performed using BD-LSRII. Expression of human HLA-DR4 wasclearly detectable on the surface of CD19+ B cells and was significantlyupregulated upon stimulation by toll-like receptor agonist poly(I:C)(see FIG. 9).

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

Entire contents of all non-patent documents, patent applications andpatents cited throughout this application are incorporated by referenceherein in their entirety.

What is claimed is:
 1. A non-human animal comprising at an endogenousMajor Histocompatibility Complex II (MHC II) α gene locus a nucleotidesequence encoding a chimeric human/non-human MHC II α polypeptide,wherein a human portion of the chimeric human/non-human MHC II αpolypeptide comprises a human MHC II α extracellular domain, and whereinthe animal expresses a functional MHC II complex on a surface of a cellof the animal.
 2. The animal of claim 1, wherein the human MHC II αextracellular domain comprises human α1 and α2 domains.
 3. The animal ofclaim 1, wherein the nucleotide sequence is expressed under regulatorycontrol of endogenous non-human MHC II α promoter and regulatoryelements.
 4. The animal of claim 1, wherein a non-human portion of thechimeric human/non-human MHC II α polypeptide comprises transmembraneand cytoplasmic domains of an endogenous non-human MHC II α polypeptide.5. The animal of claim 1, wherein the human portion of the chimerichuman/non-human MHC II α polypeptide is derived from a human HLA classII protein selected from the group consisting of HLA-DR, HLA-DQ, andHLA-DP.
 6. The animal of claim 5, wherein the human portion of thechimeric human/non-human MHC II α polypeptide is derived from a humanHLA-DR4 protein.
 7. The animal of claim 1, wherein the animal is arodent.
 8. The rodent of claim 7, wherein the rodent is a mouse.
 9. Theanimal of claim 1, wherein the animal further comprises at an endogenousMHC II β gene locus a nucleotide sequence encoding a chimerichuman/non-human MHC II β polypeptide.
 10. A non-human animal comprisingat an endogenous MHC II β gene locus a nucleotide sequence encoding achimeric human/non-human MHC II β polypeptide, wherein a human portionof the chimeric human/non-human MHC II β polypeptide comprises a humanMHC II β extracellular domain, and wherein the animal expresses afunctional MHC II complex on a surface of a cell of the animal.
 11. Theanimal of claim 10, wherein the human MHC II β extracellular domaincomprises human β1 and β2 domains.
 12. The animal of claim 10, whereinthe nucleotide sequence is expressed under regulatory control ofendogenous non-human MHC II β promoter and regulatory elements.
 13. Theanimal of claim 10, wherein a non-human portion of the chimerichuman/non-human MHC II β polypeptide comprises transmembrane andcytoplasmic domains of an endogenous non-human MHC II β polypeptide. 14.The animal of claim 10, wherein the human portion of the chimerichuman/non-human MHC II β polypeptide is derived from a human HLA classII protein selected from the group consisting of HLA-DR, HLA-DQ, andHLA-DP.
 15. The animal of claim 14, wherein the human portion of thechimeric human/non-human MHC II β polypeptide is derived from a humanHLA-DR4 protein.
 16. The animal of claim 10, wherein the animal is arodent.
 17. The rodent of claim 16, wherein the rodent is a mouse. 18.The animal of claim 10, wherein the animal further comprises at anendogenous MHC II α gene locus a nucleotide sequence encoding a chimerichuman/non-human MHC II α polypeptide.
 19. A rodent comprising at anendogenous MHC II gene locus a first nucleotide sequence encoding achimeric human/rodent MHC II α polypeptide and a second nucleotidesequence encoding a chimeric human/rodent MHC II β polypeptide, whereina human portion of the chimeric human/rodent MHC II α polypeptidecomprises a human MHC II α extracellular domain and a human portion ofthe chimeric human/rodent MHC II β polypeptide comprises a human MHC IIβ extracellular domain, and wherein the chimeric human/rodent MHC II αand MHC II β polypeptides form a functional MHC II complex on a surfaceof a cell of the rodent.
 20. The rodent of claim 19, wherein the humanMHC II α extracellular domain comprises human α1 and α2 domains.
 21. Therodent of claim 19, wherein the human MHC II β extracellular domaincomprises human β1 and β2 domains.
 22. The rodent of claim 19, whereinthe first nucleotide sequence is expressed under regulatory control ofendogenous rodent MHC II α promoter and regulatory elements, and thesecond nucleotide sequence is expressed under regulatory control ofendogenous rodent MHC II β promoter and regulatory elements.
 23. Therodent of claim 19, wherein a rodent portion of the chimerichuman/rodent MHC II α polypeptide comprises transmembrane andcytoplasmic domains of an endogenous rodent MHC II α polypeptide. 24.The rodent of claim 19, wherein a rodent portion of the chimerichuman/rodent MHC II β polypeptide comprises transmembrane andcytoplasmic domains of an endogenous rodent MHC II β polypeptide. 25.The rodent of claim 19, wherein the human portions of the chimerichuman/rodent MHC II α and β polypeptides are derived from a human HLAclass II protein selected from the group consisting of HLA-DR, HLA-DQ,and HLA-DP.
 26. The rodent of claim 25, wherein the human portions ofthe chimeric human/rodent MHC II α and β polypeptides are derived from ahuman HLA-DR4 protein.
 27. The rodent of claim 19, wherein the rodent isa mouse.
 28. The mouse of claim 27, wherein rodent portions of thechimeric MHC II α and β polypeptides are derived from a mouse H-2Eprotein.
 29. The rodent of claim 19, wherein the rodent does not expressfunctional endogenous MHC II polypeptides from their endogenous rodentMHC II loci.
 30. A mouse comprising at an endogenous mouse MHC II locusa first nucleotide sequence encoding a chimeric human/mouse MHC II αpolypeptide and a second nucleotide sequence encoding a chimerichuman/mouse MHC II β polypeptide, wherein a human portion of thechimeric MHC II α polypeptide comprises an extracellular domain derivedfrom an α polypeptide of a human HLA-DR4 protein and the human portionof the chimeric MHC II β polypeptide comprises an extracellular domainderived from a β polypeptide of a human HLA-DR4 protein, wherein a mouseportion of the chimeric MHC II α polypeptide comprises transmembrane andcytoplasmic domains of a mouse H-2E α chain and a mouse portion of thechimeric MHC II β polypeptide comprises transmembrane and cytoplasmicdomains of a mouse H-2E β chain, and wherein the mouse expresses afunctional chimeric HLA-DR4/H-2E MHC II complex on a surface of a cellof the mouse.
 31. The mouse of claim 30, wherein the extracellulardomain of the α polypeptide comprises human α1 and α2 domains.
 32. Themouse of claim 30, wherein the extracellular domain of the β polypeptidecomprises human β1 and β2 domains.
 33. The mouse of claim 30, whereinthe first nucleotide sequence is expressed under regulatory control ofendogenous mouse MHC II α promoter and regulatory elements, and thesecond nucleotide sequence is expressed under regulatory control ofendogenous mouse MHC II β promoter and regulatory elements.
 34. Themouse of claim 30, wherein the mouse does not express functionalendogenous MHC II polypeptides from their endogenous mouse loci.
 35. Amethod of modifying an MHC II locus of a mouse to express a chimerichuman/mouse MHC II complex comprising replacing at the endogenous mouseMHC II locus a nucleotide sequence encoding a mouse MHC II complex witha nucleotide sequence encoding a chimeric human/mouse MHC II complex.36. The method of claim 35, wherein the nucleotide sequence encoding thechimeric human/mouse MHC II complex comprises a first nucleotidesequence encoding an extracellular domain of a human MHC II αpolypeptide and a transmembrane and cytoplasmic domains of a mouse MHCII α polypeptide and a second nucleotide sequence encoding anextracellular domain of a human MHC II β polypeptide and transmembraneand cytoplasmic domains of a mouse MHC II β polypeptide.
 37. The methodof claim 35, wherein a human portion of the chimeric MHC II complex isderived from a human HLA class II protein selected from the groupconsisting of HLA-DR, HLA-DQ, and HLA-DP.
 38. The method of claim 35,wherein a mouse portion of the chimeric MHC II complex is derived from amouse H-2E protein, and a human portion of the chimeric MHC II complexis derived from a human HLA-DR4 protein.
 39. The method of claim 36,wherein human portions of chimeric MHC II α and β polypeptides arederived a human HLA class II protein selected from the group consistingof HLA-DR, HLA-DQ, and HLA-DP.
 40. The method of claim 36, wherein mouseportions of chimeric MHC II α and β polypeptides are derived from amouse H-2E protein, and human portions of chimeric MHC II α and βpolypeptides are derived from a human HLA-DR4 protein.
 41. The method ofclaim 35, wherein the replacement is made in a single ES cell, and thesingle ES cell is introduced into a mouse embryo to make a mouse.